The goals of this proposal are to define the role of glycosylation and of intracellular proteins in the correct folding and transport of the transferrin receptor to the cell surface. These studies will provide information concerning the mechanisms of protein transport and intra- and inter-protein signalling. The uptake of iron into mammalian cells involves the binding of transferrin, the serum iron transport protein, to the transferrin receptor, followed by the internalization of the receptor- transferrin complex. Iron uptake is essential for cell division. The receptor has been identified as a proliferation specific marker. Rapidly proliferating cells have higher iron requirements than non-dividing cells (that do not have differentiated functions for iron) and have higher numbers of these receptors. Prior studies using tunicmycin-treated A431 cells have demonstrated that the unglycosylated form of the transferrin receptor does nth form intersubunit disulfide bonds, does not bind transferrin, is not transported to the cell surface and is found associated in a complex with two proteins (Mr=130/135kd). The first aim of this proposal is to define the composition and function of the transferrin receptor-130/135kd proteins complex. Crosslinking studies will be used to determine whether other proteins are associated with this complex. The intracellular location of the unglycosylated receptor will be determined by immunohistolocalization as well as subcellular fractionation to establish whether it is associated with specific subcellular organelles. The complex will be further characterized with respect to its stability and protein interactions to determine if the 130/135kd proteins could have similarities to the family of heat shock-stress proteins. The second aim of this proposal is to study the 130/135kd proteins directly to determined their function. They will be purified and partial sequences of them will be compared with protein data bases. Antibodies will be generated against them to permit the examination and quantification of the 130/135kd proteins in untreated cells and to establish if they ar associated with the nascent transferrin receptor. The third aim is to determined the role of asparagine-linked glycosylation in the correct folding and transport of the newly synthesized receptor to the cell surface. This will be approached by using site-directed mutagenesis which will test the contribution of each of the three asparagine-linked glycosylation sites.